CVN-12p1: a recombinant allosteric lectin antagonist of HIV-1 envelope gp120 interactions

ABSTRACT

The invention provides a recombinant multi-functional chimera of CVN and 12p1. Chimeras of CVN and 12p1 present a model for targeting gp120 at two discrete sites, by two different modes of inhibition and with increasing potency versus either component alone. A chimera of the invention combines the high affinity suppression of viral activity by CVN with the allosteric suppression of viral envelope binding to both CD4 and co-receptor by 12p1.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This research was supported in part by U.S. Government funds (NIH grantnumbers P01 GM 56550 and PA-01-075) and the U.S. Government maytherefore have certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention relates to a retrovirus inhibitor and more specificallyto an HIV inhibitor.

2. Description of Related Art

There were an estimated 40.3 million people infected with HIV in 2005,with close to 5 million people becoming newly infected. AIDS isaffecting an increasing number of women worldwide particularly indeveloping countries where transmission occurs primarily throughheterosexual intercourse. A discrete, female-controlled method ofpreventing HIV infection is of paramount importance in the battleagainst AIDS. Since an effective vaccine is still years fromdevelopment, topical microbicides have gained increased attention.Topical microbicides are vaginally or rectally applied compoundsdesigned to inactivate HIV and prevent infection that should ideally beeffective, safe, inexpensive and easy to administer. Microbicides,compounds that could be used in vaginal and rectal formulations, areincreasingly seen as an urgent goal to stop transmission.

Advances in our understanding of the mechanism of HIV entry andinfection have led to the development of microbicides that can targetHIV without harming the body's natural defense system (ref 1-4). Theinitial, critical step of HIV infection is its entry through the fusionof the viral membrane with the membrane of either a T-cell ormacrophage. The fusion process is mediated by the viral envelopeglycoprotein, gp120, and can be triggered by interaction of gp120 withthe T-cell antigen receptor CD4 glycoprotein (ref 5-7). CD4 inducesconformational changes in gp120 that are postulated to promotesubsequent steps in the fusion process, such as co-receptor binding anddissociation of gp120 from p41 (ref. 8-9). Several seven-transmembranechemokine receptors, mainly CCR5 and CXR4, have been identified asobligate co-receptors for viral entry into the host cell (ref 9-13).Blocking the binding of CD4 with gp120 or preventing the CD4-inducedconformational isomerization that promotes co-receptor binding and viralcell fusion could have potential value for the prevention and treatmentof HIV infection and AIDS.

One candidate for a topical microbicide targeting gp120 iscyanovirin-N(CVN), an 11 kD protein originally isolated from thecyanobacteria Nostoc ellipsosporum (ref 14). It inactivates a broadrange of clades of HIV-1, SIV, and FIV, and prevents cell to celltransmission of infection. Recent investigations using both in vitro andin vivo assays yield support for the efficacy of CV-N as a microbicidalcandidate. Recombinant CVN blocked HIV-1 BaL infection of humanectocervical explants with no cytotoxic effects (ref 15). Gelformulations of CVN applied rectally to male macaques protected againstchallenge by the SIV/HIV-1 virus SHIV89.6P (ref 16). Furtherdemonstration of in vivo efficacy was shown in a vaginal challenge modelwith female macaques. The macaques were treated with a vaginal gelcontaining CVN and challenged with SHIV89.6P. Under the challengeconditions of this assay, all placebo-treated and untreated controls (8of 8) became infected, while 15 of 18 CVN treated macaques were notinfected. CVN showed no clinically adverse effects in these in vivoassays.

CVN binds specifically to the highly glycosylated viral envelope proteingp120 and to the functionally analogous SIV proteins sgp130 and sgp140.In contrast, CVN does not bind appreciably to the soluble form of thecellular receptor CD4 (sCD4) or to a battery of other referenceproteins. Investigations of CVN interactions by solution biophysicalmethods with both HIV-1 JRFL and HIV-1 89.6 envelope proteins gp120 andgp41 showed that the interaction of CVN with gp120 is of preferentiallyhigher affinity, in the nM K_(D) range, with a greater than 1:1stoichiometry (ref 17). The epitopes on gp120 responsible for CVNbinding appear to be predominantly high-mannose glycosylation sites ofthe Env, specifically terminal Man-α(1-2)manα-moieties on Man-8 andMan-9 glycans, and these appear key to the antagonist properties of theCVN molecule (ref 18-25). The high resolution structure of CVN has beensolved by both X-ray crystallography and nuclear magnetic resonancespectroscopy (ref 26-30). These studies have identified carbohydratebinding sites on the CVN molecule. One of these appears to be higheraffinity. Mutagenic analysis has shown that the high affinity site byitself is responsible for inhibition of HIV-1 fusion activity by CVN.Nonetheless, there are potential limitations, including amount of CVNproduction required based on measured in vivo efficacy, reliance on asingle site of action and CV-N resistant strains of virus.

Another potential microbicide is the linear peptide 12p1 which wasinitially isolated from a phage display library and found to inhibitinteraction of HIV-1 gp120 with both CD4 and a CCR5 surrogate, mAb 17b(ref 31). There is a direct interaction of 12p1 with gp120, which occurswith a binding stoichiometry of 1:1 (ref 32). The peptide was shown toinhibit the binding of monomeric YU2 gp120 to both sCD4 and 17b at IC₅₀values of 1.1 and 1.6 μM, respectively as determined by SPR analysis.This dual inhibition is a key feature of the action of 12p1. Peptide12p1 also inhibited binding of these ligands to trimeric envelopeglycoproteins, blocked the binding of gp120 to the native co-receptorCCR5, and specifically inhibited HIV-1 infection of target cells invitro. Analyses of sCD4 saturation of monomeric gp120 in the presence orabsence of a fixed concentration of peptide suggest that 12p1suppression of CD4 binding to gp120 is due to allosteric inhibitoryeffects rather than competitive inhibition of CD4 binding. Using a panelof gp120 mutants that exhibit weakened inhibition by 12p1, the putativebinding site of the peptide was mapped to a region immediately adjacentto, but distinguishable from, the CD4 binding footprint. 12p1 was unableto inhibit binding of sCD4 to a gp120 mutant, S375W, which is believedto resemble the CD4-induced conformation of gp120. The results obtainedto date strongly suggest that 12p1 preferentially binds gp120 prior toengagement of CD4, and alters the conformational state of gp120 to aform that has suppressed interactions with receptor ligands (CD4 andCCR5/CXCR4) that are generally believed crucial for viral entry.

Thus, despite the foregoing developments, there is still a need in theart for a retrovirus inhibitor and more specifically for an HIVinhibitor.

All references cited herein are incorporated herein by reference intheir entireties.

BRIEF SUMMARY OF THE INVENTION

Accordingly, in one aspect, the invention is a chimeric proteincomprising a first sequence coding for cyanovirin, a second sequencecoding for 12p1 and a linker covalently connecting the first sequencecoding for cyanovirin with the second sequence coding for 12p1, whereinthe first sequence coding for cyanovirin is selected from the groupconsisting of (a) at least nine contiguous amino acids of SEQ ID NO: 2,(b) nucleic acid sequence of SEQ ID NO: 1, (c) nucleic acid sequence ofSEQ ID NO: 3, (d) amino acid sequence of SEQ ID NO: 4, and (e) nucleicacid sequence of SEQ ID NO: 5 and the second sequence coding for 12p1 isselected from the group consisting of nucleic acid sequence of SEQ IDNO: 6 and nucleic acid sequence of SEQ ID NO: 7.

In certain embodiments, the linker comprises nucleic acid sequence ofSEQ ID NO: 8.

In certain embodiments, the linker has at least three repeats of SEQ IDNO: 8. In certain embodiments, the linker has a nucleotide sequence ofSEQ ID NO: 7.

In certain embodiments, the linker has at least five repeats of a SEQ IDNO: 8 or corresponds to SEQ ID NO: 9.

In certain embodiments, the chimeric protein has a nucleotide sequenceof SEQ ID NO: 10.

In certain embodiments, the chimeric protein has a nucleotide sequenceof SEQ ID NO: 11.

In another aspect, the invention is an HIV inhibitor comprising achimeric protein comprising a first sequence coding for cyanovirin, asecond sequence coding for 12p1 and a linker covalently connecting thefirst sequence coding for cyanovirin with the second sequence coding for12p1, wherein the first sequence coding for cyanovirin is selected fromthe group consisting of (a) at least nine contiguous amino acids of SEQID NO: 2, (b) nucleic acid sequence of SEQ ID NO: 1, (c) nucleic acidsequence of SEQ ID NO: 3, (d) amino acid sequence of SEQ ID NO: 4, and(e) nucleic acid sequence of SEQ ID NO: 5 and the second sequence codingfor 12p1 is selected from the group consisting of nucleic acid sequenceof SEQ ID NO: 6 and nucleic acid sequence of SEQ ID NO: 7.

In another aspect, the invention is a pharmaceutical compositioncomprising the HIV inhibitor as described above and a pharmaceuticallyacceptable carrier.

In another aspect, the invention is a pharmaceutical compositioncomprising:

(i) an isolated and purified first nucleic acid molecule proteinencoding a first sequence coding for cyanovirin, wherein the firstsequence coding for cyanovirin is selected from the group consisting of(a) at least nine contiguous amino acids of SEQ ID NO: 2, (b) nucleicacid sequence of SEQ ID NO: 1, (c) nucleic acid sequence of SEQ ID NO:3, (d) amino acid sequence of SEQ ID NO: 4, and (e) nucleic acidsequence of SEQ ID NO: 5; and

(ii) an isolated and purified second nucleic acid molecule proteinencoding a second sequence coding for 12p1, wherein the second sequencecoding for 12p1 is selected from the group consisting of nucleic acidsequence of SEQ ID NO: 6 and nucleic acid sequence of SEQ ID NO: 7,wherein said pharmaceutical composition is effective to prevent, treator alleviate an HIV infection in a mammal.

In another aspect, the invention is a method of preventing oralleviating an HIV infection, the method comprising providing thepharmaceutical composition comprising the HIV inhibitor as describedabove and a pharmaceutically acceptable carrier wherein saidpharmaceutical composition is provided in an amount effective toprevent, treat or alleviate an HIV infection in a mammal.

In another aspect, the invention is a method of preventing oralleviating an HIV infection, the method comprising providing thepharmaceutical composition comprising:

(i) an isolated and purified first nucleic acid molecule proteinencoding a first sequence coding for cyanovirin, wherein the firstsequence coding for cyanovirin is selected from the group consisting of(a) at least nine contiguous amino acids of SEQ ID NO: 2, (b) nucleicacid sequence of SEQ ID NO: 1, (c) nucleic acid sequence of SEQ ID NO:3, (d) amino acid sequence of SEQ ID NO: 4, and (e) nucleic acidsequence of SEQ ID NO: 5; and

(ii) an isolated and purified second nucleic acid molecule proteinencoding a second sequence coding for 12p1, wherein the second sequencecoding for 12p1 is selected from the group consisting of nucleic acidsequence of SEQ ID NO: 6 and nucleic acid sequence of SEQ ID NO: 7,wherein said pharmaceutical composition is provided in the amounteffective to prevent, treat or alleviate an HIV infection in a mammal.

In another aspect, the invention is a vector comprising the chimericprotein of the invention.

In another aspect, the invention is a host cell containing the vectorcomprising the chimeric protein of the invention.

In another aspect, the invention is a method of producing a protein,which method comprises expressing a protein in a host cell containingthe vector comprising the chimeric protein of the invention. In certainembodiments, the host cell is an autologous or a homologous mammaliancell. In certain embodiments, the host cell is a nonpathogenic bacteriumor a nonpathogenic yeast. In certain embodiments, the host cell is alactobacillus.

In another aspect, the invention is an antibody to the chimeric proteinof the invention.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

The invention will be described in conjunction with the followingdrawings in which like reference numerals designate like elements andwherein:

FIG. 1 is a graph demonstrating synergy of using 12p1 with CV-N.Inhibition effects are shown as a function of dose of CV-N, 12p1, ormixture of the two. The “mixture” data were obtained with mixtures oflowest concentration of CV-N with the lowest dose of 12p1, etc. tohighest dose CV-N with the highest dose of 12p1. The curves are alsoshown for the dose response expected if the effects were simplyadditive. These data show that mixing 12p1 with CV-N does not interferewith the latter and instead synergizes positively.

FIG. 2A shows HIV-1YU2 gp120 denoting various ligand binding sites.Potential glycosylation sites that could be sites of CV-N binding areshown as green ball and stick. The three residues found important for12p1 binding (Lys97, Glu102, and Arg476) are in space fillingrepresentations.

FIG. 2B is a schematic representation of the constructs used in thispaper (not drawn to scale). All constructs contain a pel b secretorysequence and a hexa-histidine tag for purification. The L5 chimera (SEQID NO: 10) contains the peptide 12p1 (SEQ ID NO: 6 or 7) c-terminal tothe CVN domain (SEQ ID NOs. 1-5) with an intervening linker of fiverepeats of Gly4Ser (SEQ ID NO: 9). The construct L5-S12p1 contains thenon-sense peptide (WRIMMIPSEANN) c-terminal to the CVN domain andcontains a linker of five repeats of (Gly)4Ser (SEQ ID NO: 9).

FIG. 3 is a picture of a gel demonstrating expression and purity ofchimeras. All constructs were isolated by osmotic shock and purifiedover a Nickle-NTA column. The arrowhead indicates the position of theCVN band (11 kDa) and the asterisk indicates the position of thechimerae (15 kDa). Samples were separated on a 4-20% gel under reducingconditions and stained with Simply Blue (Invitrogen) Coomassie stain.

FIG. 4A is a graph demonstrating Biosensor analysis of CVN binding toimmobilized HIV-1YU2 gp120.

FIG. 4B is a graph demonstrating Biosensor analysis of the L5 (4B)chimera binding to immobilized HIV-1YU2 gp120.

FIG. 4C are representative sensorgrams of the interaction of CVN or L5with sensor chip immobilized YU2-gp120. CVN or L5 was passed over thechip at concentrations of 300, 200, 100, 50, 25 and 12.5 nM. ELISA assayof serial dilutions of CVN (circles), L5 (squares) or L5-S12p1(triangle) were added to 100 ng of bound YU2 gp120. The extent ofbinding was detected by polyclonal anti-CVN. Points are averages oftriplicate determinations after the subtraction of non-specific bindingto BSA. Error bars indicate standard deviation.

FIG. 4D is a bar graph depicting ELISA assay of CVN or the L5 chimerabinding to gp120 of a variety of clades and tropisms. In ELISAexperiments, 100 ng of the indicated gp120 was adsorbed onto an ELISAplate followed by the addition of 50 nM of either CVN (white bars) orthe L5 chimera (black bars). The extent of binding was detected with apolyclonal antibody against CVN. Bars represent triplicate determinationafter the subtraction of non-specific binding to BSA. Error barsindicate one standard deviation.

FIGS. 5A-5B are bar graphs demonstrating inhibition of sCD4 or theco-receptor surrogate mAb 17b interaction with YU2 gp120. MonomericHIV-1YU2 gp120 was adsorbed onto ELISA plates prior to the addition ofincreasing doses of CVN, L5, L5-S12p1 or unlinked CVN and 12p1 incubatedwith either 0.5 μg/ml sCD4 (FIG. 5A) or 2 μg/ml 17b (FIG. 5B).

FIGS. 6A-6B are graphs demonstrating competition of CVN or L5 with mAbsF105 (Left) and 2G12 (Right) to gp120. Monomeric HIV-1YU2 gp120 wasadsorbed onto ELISA plates overnight prior to blocking with 1% BSA.Increasing doses of CVN (circles) or L5 (squares) was combined with a1:20,000 dilution of F105 (FIG. 6A) or 50 μg/ml 2G12 (FIG. 6B) was addedfor one hour at room temperature.

DETAILED DESCRIPTION OF THE INVENTION

This invention was prompted by the idea to form a chimeric protein entryinhibitor that combines the action two gp120-targeting molecules, anallosteric peptide inhibitor 12p1 and a higher affinitycarbohydrate-binding protein cyanovirin (CVN). The invention was drivenby a desire to develop a novel HIV inhibitor which can overcome potencylimits and potential virus mutational resistance for either 12p1 or CVNalone. Inventors created a chimeric protein which utilizes the highaffinity binding of CVN and the unique allosteric inhibition of 12p1.CVN was chosen as a fusion partner for several reasons: (i) CVN binds togp120 with high affinity and inhibits HIV from a broad range of cladesand tropisms; (ii) CVN binds to high mannose residues in regions thatdon't appear to overlap with domains traditionally targeted forinhibition as determined by its inability to compete with a variety ofmonoclonal antibodies to gp120 or sCD4; (iii) CVN is currently underinvestigation as a topical microbicide. (iv) CVN retains its antiviralability after treatment with denaturants, heat and detergents andtherefore may also be able to function after the addition of a longpolypeptide chain to one of its termini. 12p1 was chosen because (i) itinhibits in a manner distinct from CVN, (ii) is small and (iii) has nodisulfide bonds that could potentially lead to misfolding andaggregation.

The chimeric protein of the invention can be shown to inhibit a virus,specifically a retrovirus, such as the human immunodeficiency virus,i.e., HIV-1 or HIV-2. The chimeric protein of the present invention canbe used to inhibit other retroviruses as well as other viruses. Examplesof viruses that can be treated in accordance with the present inventioninclude, but are not limited to, Type C and Type D retroviruses, HTLV-1,HTLV-2, HIV, FLV, SIV, MLV, BLV, BIV, equine infectious virus, anemiavirus, avian sarcoma viruses, such as Rous sarcoma virus (RSV),hepatitis type A, B, non-A and non-B viruses, arboviruses, varicellaviruses, measles, mumps and rubella viruses

In initial mixing experiments, it was demonstrated that these inhibitorsdo not interfere with each other and show functional synergy ininhibiting viral cell infection when used together. In certainembodiments of the invention, the chimeric protein fusion inhibitorcomprises 12p1 linked to the C-terminal domain of CVN through a flexiblelinker of one through seven penta-peptide repeats of glycine and serine.The chimerae with five repeats of the linker termed, L5, binds to gp120from a variety of clades and tropisms. Advantageously, the chimeraexhibits increased inhibition of gp120 binding to receptor CD4,co-receptor surrogate mAb 17b and gp120 antibody F105. Bindinginhibition by chimera reflects both the high affinity of the CVN domainand the allosteric action of the 12p1 domain. This enhanced inhibitionis lost in constructs where the sequence of 12p1 is scrambled, renderingit inactive. This work laid a background for creating high potencychimeras, as well as non-covalent mixtures, as leads for HIV-1 envelopeantagonism that can overcome potency limits and potential virusmutational resistance for either 12p1 or CVN alone.

Chimeras of CVN and 12p1 or variants thereof present a model fortargeting gp120 at two discrete sites, by two different modes ofinhibition and with increasing potency versus either component alone. Achimera of the invention combines the high affinity suppression of viralactivity by CVN with the allosteric suppression of viral envelopebinding to both CD4 and co-receptor by 12p1. Furthermore, since thebinding sites for CVN and 12p1 both reside within gp120 but atsterically separate locations, the combination of these two agents as acovalent chimera increases the ability to overcome resistance mutationsat individual binding sites for either component alone.

In certain embodiments, CVN and 12p1 or variants thereof are covalentlylinked in a chimera to be administered in a pharmaceutical compositionto prevent, treat or alleviate HIV infection in a mammal. Thus, in oneaspect, the invention is an HIV inhibitor comprising a chimeric proteincomprising a first sequence coding for cyanovirin, a second sequencecoding for 12p1 and a linker covalently connecting the first sequencecoding for cyanovirin with the second sequence coding for 12p1, whereinthe first sequence coding for cyanovirin is selected from the groupconsisting of (a) at least nine contiguous amino acids of SEQ ID NO: 2,(b) nucleic acid sequence of SEQ ID NO: 1, (c) nucleic acid sequence ofSEQ ID NO: 3, (d) amino acid sequence of SEQ ID NO: 4, and (e) nucleicacid sequence of SEQ ID NO: 5 and the second sequence coding for 12p1 isselected from the group consisting of nucleic acid sequence of SEQ IDNO: 6 and nucleic acid sequence of SEQ ID NO: 7. In another aspect, theinvention is a pharmaceutical composition comprising the HIV inhibitoras described above and a pharmaceutically acceptable carrier.

In certain embodiments, CVN and 12p1 or variants thereof are notcovalently linked but combined as a mixture to be administered in apharmaceutical composition to prevent, treat or alleviate HIV infectionin a mammal. Thus in one aspect, the invention is a pharmaceuticalcomposition comprising: (i) an isolated and purified first nucleic acidmolecule protein encoding a first sequence coding for cyanovirin,wherein the first sequence coding for cyanovirin is selected from thegroup consisting of (a) at least nine contiguous amino acids of SEQ IDNO: 2, (b) nucleic acid sequence of SEQ ID NO: 1, (c) nucleic acidsequence of SEQ ID NO: 3, (d) amino acid sequence of SEQ ID NO: 4, and(e) nucleic acid sequence of SEQ ID NO: 5; and (ii) an isolated andpurified second nucleic acid molecule protein encoding a second sequencecoding for 12p1, wherein the second sequence coding for 12p1 is selectedfrom the group consisting of nucleic acid sequence of SEQ ID NO: 6 andnucleic acid sequence of SEQ ID NO: 7, wherein said pharmaceuticalcomposition is effective to prevent, treat or alleviate an HIV infectionin a mammal.

DEFINITIONS Cyanovirin-N

CVN is an 11 kDa protein originally isolated from the cyanobacterium,Nostic ellipsosporum (ref 14). Inactivated a broad spectrum of HIVclades including A, B, C, D and circulating recombinant forms.Cyanovirin-N specifically binds with nanomolar affinity to mammalianhigh mannose oligosaccharides, D1D3 isomer of Man8GlcNAc2 (Man8 D1D3)and Man9GlcNAc2 (Man 9). CVN is currently in development as a femalecontrolled microbicide.

The terms “cyanovirin,” “cyanovirin-N” or its abbreviation CVN is usedherein to generically refer to a native antiviral protein isolated fromNostoc ellipsosporum (“native cyanovirin”) and any functionallyequivalent protein or derivative thereof. In the context of the presentinvention, such a functionally equivalent protein or derivative thereof(a) contains a sequence of at least nine (preferably at least twenty,more preferably at least thirty, and most preferably at least fifty)amino acids directly homologous with (preferably the same as) anysubsequence of nine contiguous amino acids contained within a nativecyanovirin (especially cyanovirin-N), and (b) is antiviral, inparticular capable of specifically binding to a virus, more specificallya primate immunodeficiency virus, more specifically HIV-1, HIV-2, orSIV, or to an infected host cell expressing one or more viralantigen(s), more specifically an envelope glycoprotein, such as gp120,of the respective virus. In addition, such a functionally equivalentprotein or derivative thereof can comprise the amino acid sequence of anative cyanovirin, particularly cyanovirin-N (see SEQ ID NO: 2), inwhich 1-20, preferably 1-10, more preferably 1, 2, 3, 4, or 5, and mostpreferably 1 or 2, amino acids have been removed from one or both ends,preferably from only one end, and most preferably from theamino-terminal end, of the native cyanovirin.

Cyanovirin of the invention preferably comprises an amino acid sequencethat is substantially homologous to that of an antiviral protein fromNostoc ellipsosporum, specifically a native cyanovirin, particularlycyanovirin-N. In the context of the cyanovirins of the presentinvention, the term “substantially homologous” means sufficient homologyto render the cyanovirin antiviral, preferably with antiviral activitycharacteristic of an antiviral protein isolated from Nostocellipsosporum. There preferably exists at least about 50% homology, morepreferably at least about 75% homology, and most preferably at leastabout 90% homology.

Thus, in the present invention, a first nucleic acid sequence or acyanovarin coding sequence comprises at least one of a sequence of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

It will be apparent to one skilled in the art that a partialcyanovirin-N gene codon sequence will likely suffice to code for a fullyfunctional, i.e., antiviral, such as anti-HIV, cyanovirin. A minimumessential DNA coding sequence(s) for a functional cyanovirin can readilybe determined by one skilled in the art, for example, by synthesis andevaluation of sub-sequences comprising the native cyanovirin, and bysite-directed mutagenesis studies of the cyanovirin-N DNA codingsequence.

Using an appropriate DNA coding sequence, a recombinant cyanovirin canbe made by genetic engineering techniques (see, e.g., for generalbackground, Nicholl, in An Introduction to Genetic Engineering,Cambridge University Press: Cambridge, 1994, pp. 1-5 & 127-130). Forexample, a Nostoc ellipsosporum gene or cDNA encoding a cyanovirin canbe identified and subcloned. The gene or cDNA can then be incorporatedinto an appropriate expression vector and delivered into an appropriateprotein-synthesizing organism (e.g., E. coli, S. cerevisiae, P.pastoris, or other bacterial, yeast, insect, or mammalian cell), wherethe gene, under the control of an endogenous or exogenous promoter, canbe appropriately transcribed and translated. Such expression vectors(including, but not limited to, phage, cosmid, viral, and plasmidvectors) are known to those skilled in the art, as are reagents andtechniques appropriate for gene transfer (e.g., transfection,electroporation, transduction, micro-injection, transformation, etc.).Subsequently, the recombinantly produced protein can be isolated andpurified using standard techniques known in the art (e.g.,chromatography, centrifugation, differential solubility, isoelectricfocusing, etc.), and assayed for antiviral activity.

Alternatively, a native cyanovirin can be obtained from Nostocellipsosporum by non-recombinant methods and sequenced by conventionaltechniques. The sequence can then be used to synthesize thecorresponding DNA, which can be subcloned into an appropriate expressionvector and delivered into a protein-producing cell for en massrecombinant production of the desired protein.

Peptide 12p1

12p1 is a linear peptide, RINNIPWSEAMM (SEQ ID NO: 6) which wasdiscovered by a phage display library (ref 31). 12p1 has the followingstructure:

12p1 inhibits CD4, 17b co-receptor surrogate, and CCR5 interaction bybinding to monomeric and trimeric gp120. Binding site mapping viamAb-binding assays indicated that 12p1 has different effects uponbinding of gp120 to conformational and non-conformationally dependentantibodies. This hints towards the binding site being present in aparticular gp120 conformational state. 12p1's mode of inhibition appearsto be allosteric in nature, as inferred from the inability of excesssCD4 to overcome the inhibitory effect of a set concentration of thepeptide.

Variants of 12p1 would include a sequence consisting of I N N I P W S(SEQ. ID NO: 7).

Possible options for useful chemical modifications of a cyanovirin, 12p1or a chimera prepared from these compounds include, but are not limitedto, (a) olefin substitution, (b) carbonyl reduction, (c) D-amino acidsubstitution, (d) N alpha-methyl substitution, (e) C alpha-methylsubstitution, (f) C alpha-C′-methylene insertion, (g) dehydro amino acidinsertion, (h) retro-inverso modification, (i) N-terminal to C-terminalcyclization, and (j) thiomethylene modification. Cyanovirins, 12p1 or achimera prepared from these compounds also can be modified by covalentattachment of carbohydrate and polyoxyethylene derivatives, which areexpected to enhance stability and resistance to proteolysis (Abuchowskiet al., in Enzymes as Drugs, Holcenberg et al., eds., John Wiley: NewYork, 1981, pp. 367-378).

Linker

Since the precise binding sites for CVN are not known, inventors couldnot estimate the distance between the binding sites of the two compoundsin the known high resolution structure of gp120 (FIG. 2A). A furthercomplication was the predicted 5:1 binding ratio of CVN, unlike the 1:1binding ratio of 12p1. In one embodiment, the linker is a 25 amino acidlinker which acts as a starting inter-domain spacer (SEQ ID NO: 9). Theamino acid composition of the linkers is also an important parameter toconsider since it will determine secondary structural characteristics ofthe spacer. The linker useful in this invention preferably contains acombination of glycine and serine residues which provides flexibilityand protease resistance. Rigidity of the linker can also vary.

In a preferred embodiment, the linker comprises aminoacids which induceflexible conformation. For example, the linker can have one to sevenpenta-peptide repeats of glycine and serine (SEQ ID NO: 8). In otherembodiments of the invention, the linker can contain amino acids thatinduce a rigid conformation. In other embodiments, the linker can be apolymer.

Chimeric Proteins of the Invention

In one aspect, the invention is a chimeric protein comprising a firstsequence coding for cyanovirin, a second sequence coding for 12p1 and alinker covalently connecting the first sequence coding for cyanovirinwith the second sequence coding for 12p1, wherein the first sequencecoding for cyanovirin is selected from the group consisting of (a) atleast nine contiguous amino acids of SEQ ID NO: 2, (b) nucleic acidsequence of SEQ ID NO: 1, (c) nucleic acid sequence of SEQ ID NO: 3, (d)amino acid sequence of SEQ ID NO: 4, and (e) nucleic acid sequence ofSEQ ID NO: 5 and the second sequence coding for 12p1 is selected fromthe group consisting of nucleic acid sequence of SEQ ID NO: 6 andnucleic acid sequence of SEQ ID NO: 7. The chimeric protein of theinvention comprises 12p1 linked to the C-terminal domain of cyanovirinvia a linker prepared by methods known in the art.

In a preferred embodiment of the invention, the chimera is a construct(SEQ. ID NO: 10) in which the N-terminal domain of 12p1 (SEQ ID NO: 6)is linked to the C-terminal domain of CVN (SEQ ID NO: 5) via a longflexible linker of five Gly₄Ser repeats (FIG. 2B) (SEQ. ID NO: 9).

In certain embodiments, the chimera, designated L5, contains ahexa-histidine tag on its C-terminus both for ease of purification andfor confirming the accessibility of the C-terminal 12p1 domain (SEQ IDNO: 11)

Also designed was a chimera in which the 12p1 sequence was scrambled,rendering it non-functional (L5-S12p1). The latter construct was derivedfrom the L5 chimera and was identical to it in all aspects exceptbinding functionality.

CVN, L5 and L5-S12p1 were expressed in E. coli using the inductionconditions optimized in Colleluori et al (ref 37). All constructs wereexpressed in the periplasmic fraction, isolated by osmotic shock andpurified over a NiNTA column. The similar high degree of homogeneity ofall purified expressed proteins was determined by Coomassie stain (FIG.3) and western blot.

The constructs can be cloned into the pET30b+prokaryotic expressionsystem and transformed into BL21 (DE3) strain of E. coli.

Methods of making the chimeric protein of the invention include:

(a) providing a first sequence coding for cyanovirin and a secondsequence coding for 12p1 and a linker, wherein the first sequence codingfor cyanovirin is selected from the group consisting of (a) at leastnine contiguous amino acids of SEQ ID NO: 2, (b) nucleic acid sequenceof SEQ ID NO: 1, (c) nucleic acid sequence of SEQ ID NO: 3, (d) aminoacid sequence of SEQ ID NO: 4, and (e) nucleic acid sequence of SEQ IDNO: 5 and the second sequence coding for 12p1 is selected from the groupconsisting of nucleic acid sequence of SEQ ID NO: 6 and nucleic acidsequence of SEQ ID NO: 7.

(b) ligating the linker and the second nucleic acid sequence (a 12p1coding sequence) to a C-terminal in the first sequence coding forcyanovirin to form a vector comprising the chimeric protein;

(c) transforming the vector into appropriate cells (e.g., BL21 (DE3))and grow until the absorbance at 600 reach a desired value (e.g., 1.2)and optionally induce the culture (e.g., 1 mM IPTG for 2 hours); and

(d) isolating the chimeric protein from the periplasmic space usingosmotic shock and purified over nickel NTA agarose column to obtain thedesired chimeric protein in substantially pure form.

In the preferred embodiment, the L5 chimera was constructed usingstandard recombinant DNA techniques. CVN was amplified from a plasmidtemplate using the forward primer 5′pelb and the reverse primer 3CVN(described in Table 1) in order to insert a pel b secretory sequence andeliminate the stop codon. CVN was ligated into pET30b+vector generatingpCVN. The linker, 12p1 (or scrambled 12p1) cassettes were synthesized asseparate oligonucleotides (Invitrogen) and ligated in frame with theC-terminal domain of CVN generating pL5 (or pL5-S12p1). The entireconstruct was amplified with the forward primer 5′pelb and a reverseprimer containing a hexa-histidine tag (3′12p1His) generating pL5H is.The construct was sequenced by DNA Sequencing Facility prior totransformation into BL21 (DE3) strain of Eschericia coli.

L5 was stable over prolonged periods and multiple freeze thaw cycles.The chimera was found to bind to gp120 with similar affinity to CVNalone as determined by both ELISA and Biacore assays (FIGS. 4A-D). Thisindicates that CVN is able to tolerate the addition of a longpolypeptide tail onto its C-terminal domain with no disruption of itsability to bind gp120 with high affinity. While we did not expect anddid not observe an enhancing effect of the 12p1 domain on the overallgp120 binding affinity of the chimera due to the substantially weakerbinding affinity of 12p1, the chimera nonetheless exhibited importantnew binding properties to the HIV-1 Env. In particular, the chimera wasable to exhibit the combined interaction-inhibiting activities unique tothe 12p1 domain with the much stronger affinity and slow dissociationrate of the CVN domain. This was evident in the inhibition of binding toCD4, 17b and F105 (FIGS. 5, 6). This pattern of inhibition is lost whenthe 12p1 domain is scrambled. These observations demonstrate that thetwo domains of the chimera can bind at the same time to gp120, with theresult of a high affinity, allosteric inhibitor that has the potentialto suppress both host cell receptor interactions of the viral envelope.The wide specificity of the CVN moiety (FIGS. 4A-D) suggests that thechimera has potential usefulness across diverse variants of the HIV-1virus. Broad specificity has recently been observed for variants of 12p1peptide (Cocklin S. et al (2007) J. Virol. 81(7):3645), reinforcingfurther the opportunity to employ the antagonist potential of thechimera to inhibit diverse viral forms, even those in which one of thetarget epitopes (either carbohydrate for CVN or binding footprint for12p1) is mutated. Direct binding of CVN or the chimera to YU2 variant ofgp120

Inventors initially confirmed that the L5 chimera retains the highaffinity binding to gp120 characteristic of the CVN domain through bothELISA and biosensor analysis. The binding to CVN or the L5 chimera toimmobilized gp120 was analyzed on a Biacore 3000 SPR optical biosensor.Approximately 250 RUs of either YU2 gp120 or the non-specific controlantibody 2B6R were immobilized to a CM5 dextran chip by amine coupling.Either CVN or L5 were passed over the chip at a flow rate of 30 μl/minfor one minute association; the dissociation phase was for two minutesat the same flow rate. Signals from the reference 2B6R flow cell wereused to correct for non-specific interactions and bulk effects (FIGS.4A, B). Biosensor response is expected to be proportional to molecularweight, with a maximum response of 33 RU for CVN binding and 45 RU forL5 binding assuming 1:1 binding stoichiometry. CVN was found to bindwith a 95 RU signal at 300 nM indicating a 3:1 stoichiometry, while L5bound with 90 RU signal at 300 nM indicative of a 2:1 stoichiometry.While these stoichiometries do not approach the 5:1 value previouslyreported for CVN, the concentrations used in this study were below thesaturation levels for each complex to prevent non-specific aggregationcommonly seen at elevated levels. The results suggest that both CVN andL5 bind to gp120 multivalently, and that L5 binds at a somewhat lowerstoichiometry than CVN.

ELISA assays were also performed to detect the binding of CVN or thechimera to YU2 gp120 (FIG. 4C). YU2 was adsorbed to a 96-well EIA/RIAplate overnight at 4° C. The wells were blocked and washed prior to theaddition of serial dilutions of CVN, L5 or L5-S 12p1 for one hour atroom temperature. The extent of binding was determined by a polyclonalantibody directed against CVN. The chimera retained its ability to bindto gp120 at concentrations equivalent to that of unmodified CVN. Thisindicates that the addition of 37 amino acids onto the c-terminal domainof CVN does not alter its ability to bind to gp120, supporting itspotential as for fusion inhibition. The equilibrium dissociationconstant K_(D) for binding of CVN to YU2 has been reported as 11 nM, ascompared to 3.6 μM for 12p1³⁸. Based on these affinity values, one wouldexpect that binding of the chimera to gp120 would be primarily directedby the CVN domain of the chimera.

CVN has the ability to inhibit X4-tropic, R5-tropic and dual tropicprimary isolates and laboratory adapted strains of HIV^(20,44). Wetherefore tested the ability of the chimera to bind to the gp120s from avariety of strains of HIV-1 of diverse clades and tropisms. Theseincluded HIV-1_(SF162) (Clade B), HIV-1_(93MW959) (Clade C),HIV-1_(92UG21-9) (Clade A), HIV-1_(92US715) (Clade B), SIV_(PBj2-8) fromJim Arthos at NIAID, AN1 (an ancestral clade B HIV provided by JimMullins⁴⁵, HIV-1_(BaL) (clade B), HIV-1_(CM235) (circulating recombinantCRF01-AE obtained from Protein Sciences, Corporation via the NIAID AIDSReference and Reagent Program). Both the L5 chimera and CVN behavedequivalently with all strains tested (FIG. 4D). The diverse HIV-1variant specificity hints at their potential for the development of atopical microbicide against stains of HIV that afflict sub-SaharanAfrica and other areas of high AIDS occurrence.

Viral Synergy Analysis

Although it is known that CVN binds to high mannose groups on theheavily glycosylated gp120, the specific binding site is unknown.Therefore, inventors initially screened for the ability of CVN and 12p1to bind to gp120 simultaneously and inhibit viral infection withoutinterfering with each other. These assays were performed by theincubation of the indicated doses of CVN and 12p1 alone or incombination with HIV-1 BaL for one hour prior to the addition of PM-1cells. Viral replication was determined by gp120 ELISA after a seven dayincubation. Dose response curves from the mean of three independentexperiments are given in FIG. 1. From these and the additional data, asynergy index for the combination was found to be in the 0.3-0.5 range,compared to a scale of 0.1-0.3 for strong synergism, 0.3-0.7 forsynergism, 0.7-0.85 for moderate synergism, 0.85-0.9 for slightsynergism and 1.10-1.20 for nearly additive. Even though the synergismshown by these results is modest, it must be borne in mind that CVN hasa far greater affinity than 12p1, therefore any effect in non-covalentmixtures using 12p1 is significant. Graphical evidence for how 12p1 canboost the activity of CVN can be seen in FIG. 1 left by comparing thevalues of % inhibition observed for CVN at the two lowest concentrationseither alone (closed triangles) or with CVN at the same two lowestconcentrations but with the two lowest concentrations of 12p1 (16-31 μM)added (open triangles). Even though 12p1 at those latter concentrationsshows only small inhibitory effect on its own, the presence of 12p1 withCVN significantly boosts efficacy in the mixture by a factor of close to2-fold. These results indicate that not only can CVN and 12p1 bind gp120simultaneously but also appear to act to synergistically enhance viralinhibition.

CVN inhibits the interaction of cell associated CD4 with gp120 but haslimited effect upon the interaction between soluble CD4 (sCD4) andgp120^(46,47). In contrast, 12p1 can inhibit the interaction of sCD4with gp120 with an IC₅₀ value of 1.1 μM³⁸. The ability of the L5 chimerato inhibit CD4 binding to gp120 was investigated. YU2 gp120 wasimmobilized onto a 96 well ELISA plate overnight. The plate was blocked,and increasing doses of CVN, L5 or L5-S12p1 combined with 0.5 μg/ml sCD4were added. The extent of binding was detected with a polyclonalantibody against CD4. The data are represented as percent of uninhibitedafter non-specific binding to BSA was subtracted out (FIG. 5A). The datademonstrate that the L5 chimera can inhibit CD4-gp120 interaction morepotently than CVN alone. This enhanced inhibition is lost when the 12p1domain of the chimera is scrambled.

The monoclonal antibody 17b binds to a CD4 induced conformation and canbe used as a surrogate for co-receptor binding^(48,49) CVN cannotinhibit this interaction⁴⁶ while 12p1 inhibits the interaction with anIC₅₀ value of 1.6 μM³⁸, The ability of the L5 chimera to inhibit thisinteraction was determined through an ELISA. The L5 chimera couldinhibit the interaction between 17b and gp120 with an IC₅₀ value of 38nM while neither CVN nor L5-S12p1 had any effect (FIG. 5B). Theinability of the L5-S12p1 chimera to inhibit this interaction indicatesthat the greatly enhanced 17b binding antagonism function is impartedspecifically by the 12p1 domain and is not due to either a contaminantor non-specific interaction of the linker domain. Strikingly, thisinhibition occurs at a dose that reflects the high affinity binding ofthe CVN domain of the chimera. The L5 chimera inhibits both the sCD4 and17b interaction with gp120 at doses at which neither CVN nor 12p1 has aneffect. These results demonstrate that both domains of the chimera areable to bind gp120 simultaneously. The chimera has the unique inhibitoryproperties of 12p1 with the high affinity binding of CVN.

Inhibition by CVN or the Chimera with mAb F105 and 2G12

An additional and clear-cut measure of 12p1's binding function in thechimera was determined, as shown in FIGS. 6A-B, by the ability of thechimera to inhibit gp120 binding to the monoclonal antibody F105⁵⁰⁻⁵².This antibody recognizes the conformationally-flexible, CD4-unligandedform of gp120⁵³. CVN has minimal effect on F105 binding to gp120⁴⁶ while12p1 inhibits the F105-gp120 interaction³⁸. mAb 2G12 was used as anegative control, since previous data have shown³⁸ that binding of 12p1to gp120 would not be expected to affect binding to this antibody.Strikingly, the suppression of F105 binding was found to occur at nMconcentrations, in contrast to unlinked 12p1 which inhibits at μMconcentrations⁴⁶ or CVN which has a more muted effect on F105 binding togp120⁵⁴. This dose-response behavior thus reflects both the highaffinity binding of CVN to gp 120 and the inhibitory effect of 12p1. Incontrast, the chimera as well as CVN show the expected competition ofgp120 binding to immobilized 2G12 antibody, the latter of which binds tocarbohydrate moieties similar to sites for CVN⁴⁶.

In another aspect, the invention is a method of preventing oralleviating an HIV infection, the method comprising providing thepharmaceutical composition comprising the HIV inhibitor as describedabove and a pharmaceutically acceptable carrier wherein saidpharmaceutical composition is provided in an amount effective toprevent, treat or alleviate an HIV infection in a mammal.

In another aspect, the invention is a method of preventing oralleviating an HIV infection, the method comprising providing thepharmaceutical composition comprising:

(i) an isolated and purified first nucleic acid molecule proteinencoding a first sequence coding for cyanovirin, wherein the firstsequence coding for cyanovirin is selected from the group consisting of(a) at least nine contiguous amino acids of SEQ ID NO: 2, (b) nucleicacid sequence of SEQ ID NO: 1, (c) nucleic acid sequence of SEQ ID NO:3, (d) amino acid sequence of SEQ ID NO: 4, and (e) nucleic acidsequence of SEQ ID NO: 5; and

(ii) an isolated and purified second nucleic acid molecule proteinencoding a second sequence coding for 12p1, wherein the second sequencecoding for 12p1 is selected from the group consisting of nucleic acidsequence of SEQ ID NO: 6 and nucleic acid sequence of SEQ ID NO: 7,wherein said pharmaceutical composition is provided in the amounteffective to prevent, treat or alleviate an HIV infection in a mammal.

The appropriate delivery system for a given chimeric protein will dependupon its particular nature, the particular clinical application, and thesite of drug action. As with any protein drug, oral delivery of achimeric protein of the invention will likely present special problems,due primarily to instability in the gastrointestinal tract and poorabsorption and bioavailability of intact, bioactive drug therefrom.Therefore, especially in the case of oral delivery, but also possibly inconjunction with other routes of delivery, it will be necessary to usean absorption-enhancing agent in combination with a given chimericprotein of the invention. A wide variety of absorption-enhancing agentshave been investigated and/or applied in combination with protein drugsfor oral delivery and for delivery by other routes (Verhoef, 1990,supra; van Hoogdalem, Pharmac. Ther. 44, 407-443, 1989; Davis, J. Pharm.Pharmacol. 44(Suppl. 1), 186-190, 1992). Most commonly, typicalenhancers fall into the general categories of (a) chelators, such asEDTA, salicylates, and N-acyl derivatives of collagen, (b) surfactants,such as lauryl sulfate and polyoxyethylene-9-lauryl ether, (c) bilesalts, such as glycholate and taurocholate, and derivatives, such astaurodihydrofusidate, (d) fatty acids, such as oleic acid and capricacid, and their derivatives, such as acylcarnitines, monoglycerides, anddiglycerides, (e) non-surfactants, such as unsaturated cyclic ureas, (f)saponins, (g) cyclodextrins, and (h) phospholipids.

Other approaches to enhancing oral delivery of protein drugs, such asthe chimeric protein of the present invention, can include theaforementioned chemical modifications to enhance stability togastrointestinal enzymes and/or increased lipophilicity. Alternatively,the protein drug can be administered in combination with other drugs orsubstances which directly inhibit proteases and/or other potentialsources of enzymatic degradation of proteins. Yet another alternativeapproach to prevent or delay gastrointestinal absorption of proteindrugs, such as the chimeric proteins of the invention, is to incorporatethem into a delivery system that is designed to protect the protein fromcontact with the proteolytic enzymes in the intestinal lumen and torelease the intact protein only upon reaching an area favorable for itsabsorption. A more specific example of this strategy is the use ofbiodegradable microcapsules or microspheres, both to protect vulnerabledrugs from degradation, as well as to effect a prolonged release ofactive drug (Deasy, in Microencapsulation and Related Processes,Swarbrick, ed., Marcell Dekker, Inc.: New York, 1984, pp. 1-60, 88-89,208-211). Microcapsules also can provide a useful way to effect aprolonged delivery of a protein drug, such as a cyanovirin or conjugatethereof, after injection (Maulding, J. Controlled Release 6, 167-176,1987).

Given the aforementioned potential complexities of successful oraldelivery of a protein drug, it is preferred in many situations that thechimeric protein of the invention be delivered by one of the numerousother potential routes of delivery of a protein drug. These routesinclude intravenous, intraarterial, intrathecal, intracisternal, buccal,rectal, nasal, pulmonary, transdermal, vaginal, ocular, and the like(Eppstein, 1988, supra; Siddiqui et al., 1987, supra; Banga et al.,1988, supra; Sanders, 1990, supra; Verhoef, 1990, supra; Barry, inDelivery Systems for Peptide Drugs, Davis et al., eds., Plenum Press:New York, 1986, pp. 265-275; Patton et al., Adv. Drug Delivery Rev. 8,179-196, 1992). With any of these routes, or, indeed, with any otherroute of administration or application, a protein drug, such as acyanovirin or conjugate thereof, may initiate an immunogenic reaction.In such situations it may be necessary to modify the molecule in orderto mask immunogenic groups. It also can be possible to protect againstundesired immune responses by judicious choice of method of formulationand/or administration. For example, site-specific delivery can beemployed, as well as masking of recognition sites from the immune systemby use or attachment of a so-called tolerogen, such as polyethyleneglycol, dextran, albumin, and the like (Abuchowski et al., 1981, supra;Abuchowski et al., J. Biol. Chem. 252, 3578-3581, 1977; Lisi et al., J.Appl. Biochem. 4, 19-33, 1982; Wileman et al., J. Pharm. Pharmacol. 38,264-271, 1986). Such modifications also can have advantageous effects onstability and half-life both in vivo and ex vivo. Other strategies toavoid untoward immune reactions also can include the induction oftolerance by administration initially of only low doses. In any event,it will be apparent from the present disclosure to one skilled in theart that for any particular desired medical application or use ofchimeric protein of the invention, the skilled artisan can select fromany of a wide variety of possible compositions, routes ofadministration, or sites of application, whatever is advantageous.

Accordingly, the chimeric protein of the present invention can beformulated into various compositions for use either in therapeutictreatment methods for virally, e.g., HIV, infected individuals, or inprophylactic methods against viral, e.g., HIV, infection of uninfectedindividuals.

Thus, the present invention provides a composition comprising thepresent inventive cyanovirin or cyanovirin conjugate, especially apharmaceutical composition comprising an antiviral effective amount ofan isolated and purified cyanovirin or cyanovirin conjugate and apharmaceutically acceptable carrier. Instead of, or in addition to, theaforementioned isolated and purified chimeric protein of the invention,the composition can comprise viable host cells transformed to directlyexpress a cyanovirin or conjugate thereof in vivo. The compositionfurther can comprise an antiviral effective amount of at least oneadditional antiviral compound other than the chimeric protein of theinvention. Suitable antiviral compounds include AZT, ddI, ddC,gancyclovir, fluorinated dideoxynucleosides, nevirapine, R82913, Ro31-8959, BI-RJ-70, acyclovir, .alpha.-interferon, recombinant sCD4,michellamines, calanolides, nonoxynol-9, gossypol and derivativesthereof, and gramicidin. The chimeric protein of the invention used inthe pharmaceutical composition can be isolated and purified fromnaturally occurring organisms or from genetically engineered organisms.Similarly, cyanovirin conjugates can be derived from geneticallyengineered organisms or from chemical coupling.

The present inventive compositions can be used to treat a virallyinfected animal, such as a human. The compositions of the presentinvention are particularly useful for inhibiting the growth orreplication of a virus, such as a retrovirus, in particular a humanimmunodeficiency virus, specifically HIV-1 and HIV-2. The compositionsare useful in the therapeutic or prophylactic treatment of animals, suchas humans, who are infected with a virus or who are at risk for viralinfection, respectively. The compositions also can be used to treatobjects or materials, such as medical equipment, supplies, or fluids,including biological fluids, such as blood, blood products, and tissues,to prevent viral infection of an animal, such as a human. Suchcompositions also are useful to prevent sexual transmission of viralinfections, e.g., HIV, which is the primary way in which the world'sAIDS cases are contracted (Merson, 1993, supra).

Potential virucides used or being considered for application againstsexual transmission of HIV are very limited; present agents in thiscategory include, for example, nonoxynol-9 (Bird, AIDS 5, 791-796,1991), gossypol and derivatives (Polsky et al., Contraception 39,579-587, 1989; Lin, Antimicrob. Agents Chemother. 33, 2149-2151, 1989;Royer, Pharmacol. Res. 24, 407-412, 1991), and gramicidin (Bourinbair,Life Sci./Pharmacol. Lett. 54, PL5-9, 1994; Bourinbair et al.,Contraception 49, 131-137, 1994).

One skilled in the art will appreciate that various routes ofadministering a drug are available, and, although more than one routemay be used to administer a particular drug, a particular route mayprovide a more immediate and more effective response than by anotherroute. Furthermore, one skilled in the art will appreciate that theparticular pharmaceutical carrier employed will depend, in part, uponthe particular cyanovirin, conjugate thereof, or host cell employed, andthe chosen route of administration. Accordingly, there is a wide varietyof suitable formulations of the composition of the present invention.

Formulations suitable for oral, rectal, or vaginal administration canconsist of, for example, (a) liquid solutions or suspensions, such as aneffective amount of the pure compound(s), and/or host cell(s) engineeredto produce the a chimeric protein of the invention, dissolved orsuspended in diluents, such as water, culture medium, or saline, (b)capsules, suppositories, sachets, tablets, lozenges, or pastilles, eachcontaining a predetermined amount of the active ingredient(s), assolids, granules, or freeze-dried cells, and (c) oil-in-water emulsionsor water-in-oil emulsions. Tablet forms can include one or more oflactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible carriers. Lozengescan comprise the active ingredient in a flavor, for example sucrose andacacia or tragacanth, while pastilles can comprise the active ingredientin an inert base, such as gelatin and glycerin, or sucrose and acacia.Suitable formulations for oral or rectal delivery also can beincorporated into synthetic and natural polymeric microspheres, or othermeans to protect the agents of the present invention from degradationwithin the gastrointestinal tract (see, for example, Wallace et al.,Science 260, 912-915, 1993). Formulations for rectal or vaginaladministration can be presented as a suppository with a suitable aqueousor nonaqueous base; the latter can comprise, for example, cocoa butteror a salicylate. Furthermore, formulations suitable for vaginaladministration can be presented as pessaries, suppositories, tampons,creams, gels, pastes, foams, or spray formulas containing, in additionto the active ingredient, such as, for example, freeze-driedlactobacilli genetically engineered to directly produce a chimericprotein of the present invention, such carriers as are known in the artto be appropriate. Similarly, the active ingredient can be combined witha lubricant as a coating on a condom.

For in vivo uses, the dose of a chimeric protein of the invention, hostcells producing a chimeric protein of the invention, or compositionthereof, administered to an animal, particularly a human, in the contextof the present invention should be sufficient to effect a prophylacticand/or therapeutic response in the individual over a reasonabletime-frame. The dose used to achieve a desired virucidal concentrationin vivo (e.g., 0.1-1000 nM) will be determined by the potency of theparticular chimeric protein of the invention, the severity of thedisease state of infected individuals, as well as, in the case ofsystemic administration, the body weight and age of the infectedindividual. The effective or virucidal dose also will be determined bythe existence of any adverse side-effects that may accompany theadministration of the particular chimeric protein of the inventionemployed. It is always desirable, whenever possible, to keep adverseside effects to a minimum.

The dosage can be in unit dosage form, such as a tablet or capsule. Theterm “unit dosage form” as used herein refers to physically discreteunits suitable as unitary dosages for human and animal subjects, eachunit containing a predetermined quantity of a chimeric protein of theinvention, alone or in combination with other antiviral agents,calculated in a quantity sufficient to produce the desired effect inassociation with a pharmaceutically acceptable carrier, diluent, orvehicle.

The specifications for the unit dosage forms of the present inventiondepend on the particular chimeric protein of the invention employed, andthe effect to be achieved, as well as the pharmacodynamics associatedwith each chimeric protein of the invention, host cells, or compositionthereof in the treated animal. The dose administered should be an“anitiviral effective amount” or “virucidal effective amount” or anamount necessary to achieve an “effective virucidal level” in theindividual animal, e.g., the human patient.

Since the “effective virucidal level” is used as the preferred endpointfor dosing, the actual dose and schedule can vary, depending uponinterindividual differences in pharmacokinetics, drug distribution, andmetabolism. The “effective virucidal level” can be defined, for example,as the blood or tissue level (e.g., 0.1-1000 nM) desired in the patientthat corresponds to a concentration of one or more chimeric protein ofthe invention, which inhibits a virus, such as HIV-1 and/or HIV-2, in anassay known to predict for clinical antiviral activity of chemicalcompounds and biological agents. The “effective virucidal level” foragents of the present invention also can vary when the chimeric proteinof the invention, is used in combination with AZT or other knownantiviral compounds or combinations thereof.

One skilled in the art can easily determine the appropriate dose,schedule, and method of administration for the exact formulation of thecomposition being used, in order to achieve the desired “effectivevirucidal level” in the individual patient. One skilled in the art alsocan readily determine and use an appropriate indicator of the “effectorconcentration” of the compounds of the present invention by a direct(e.g., analytical chemical analysis) or indirect (e.g., with surrogateindicators such as p24 or RT) analysis of appropriate patient samples(e.g., blood and/or tissues).

In the treatment of some virally infected individuals, it may bedesirable to utilize a “mega-dosing” regimen, wherein a large dose of aselected chimeric protein of the invention is administered, and timethereafter is allowed for the drug to act, and then a suitable reagentis administered to the individual to inactivate the drug.

The pharmaceutical composition can contain other pharmaceuticals, inconjunction with the chimeric protein of the invention, or host cellsproducing the chimeric protein of the invention, when used totherapeutically treat a viral infection, such as that which causes AIDS.Representative examples of these additional pharmaceuticals includeantiviral compounds, virucides, immunomodulators, immunostimulants,antibiotics, and absorption enhancers. Exemplary antiviral compoundsinclude AZT, ddI, ddC, gancylclovir, fluorinated dideoxynucleosides,nonnucleoside analog compounds, such as nevirapine (Shih et al., PNAS88, 9878-9882, 1991), TIBO derivatives, such as R82913 (White et al.,Antiviral Res. 16, 257-266, 1991), BI-RJ-70 (Merigan, Am. J. Med. 90(Suppl.4A), 8S-17S, 1991), michellamines (Boyd et al., J. Med. Chem. 37,1740-1745, 1994), and calanolides (Kashman et al., J. Med. Chem. 35,2735-2743, 1992), nonoxynol-9, gossypol and derivatives, and gramicidin(Bourinbair et al., 1994, supra). Exemplary immunomodulators andimmunostimulants include various interleukins, sCD4, cytokines, antibodypreparations, blood transfusions, and cell transfusions. Exemplaryantibiotics include antifungal agents, antibacterial agents, andanti-Pneumocystitis carnii agents. Exemplary absorption enhancersinclude bile salts and other surfactants, saponins, cyclodextrins, andphospholipids.

In a preferred embodiment of the present invention, a method offemale-controllable prophylaxis against HIV infection comprises theintravaginal administration and/or establishment of, in a female human,a persistent intravaginal population of lactobacilli that have beentransformed with a coding sequence of the chimeric protein of thepresent invention to produce, over a prolonged time, effective virucidallevels of a cyanovirin or conjugate thereof, directly on or within thevaginal and/or cervical and/or uterine mucosa.

In another aspect, the invention is a vector comprising the chimericprotein of the invention. In another aspect, the invention is a hostcell containing the vector comprising the chimeric protein of theinvention.

It also will be appreciated by one skilled in the art that a DNAsequence of the chimeric protein of the invention can be inserted exvivo into mammalian cells previously removed from a given animal, inparticular a human. Such transformed autologous or homologous hostcells, reintroduced into the animal or human, will express directly thecorresponding chimeric protein of the invention in vivo. The feasibilityof such a therapeutic strategy to deliver a therapeutic amount of anagent in close proximity to the desired target cells and pathogens(e.g., to the virus, more particularly to the retrovirus, specificallyto HIV and its envelope glycoprotein gp120), has been demonstrated instudies with cells engineered ex vivo to express sCD4 (Morgan et al.,1994, supra). As an alternative to ex vivo insertion of the DNAsequences of the present invention, such sequences can be inserted intocells directly in vivo, such as by use of an appropriate viral or othersuitable vector. Such cells transfected in vivo may be expected toproduce antiviral amounts of the chimeric protein directly in vivo.

Given the present disclosure, it will be additionally appreciated that aDNA sequence corresponding to a chimeric protein of the invention can beinserted into suitable nonmammalian host cells, and that such host cellswill express therapeutic or prophylactic amounts of the chimeric proteindirectly in vivo within a desired body compartment of an animal, inparticular a human.

In another aspect, the invention is a method of producing a protein,which method comprises expressing a protein in a host cell. In certainembodiments, the host cell is an autologous or a homologous mammaliancell. In certain embodiments, the host cell is a nonpathogenic bacteriumor a nonpathogenic yeast. In certain embodiments, the host cell is alactobacillus.

The present invention also provides antibodies directed to the chimericprotein of the invention. The availability of antibodies to any givenprotein is highly advantageous, as it provides the basis for a widevariety of qualitative and quantitative analytical methods, separationand purification methods, and other useful applications directed to thesubject proteins. Accordingly, given the present disclosure and theproteins of the present invention, it will be readily apparent to oneskilled in the art that antibodies, in particular antibodiesspecifically binding to a protein of the present invention, can beprepared using well-established methodologies (e.g., such as themethodologies described in detail by Harlow and Lane, in Antibodies. ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,1988, pp. 1-725). Such antibodies can comprise both polyclonal andmonoclonal antibodies. Furthermore, such antibodies can be obtained andemployed either in solution-phase or coupled to a desired solid-phasematrix. Having in hand such antibodies as provided by the presentinvention, one skilled in the art will further appreciate that suchantibodies, in conjunction with well-established procedures (e.g., suchas described by Harlow and Lane (1988, supra) comprise useful methodsfor the detection, quantification, or purification of a chimeric proteinof the invention, or host cell transformed to produce a chimeric proteinof the invention.

The invention will be illustrated in more detail with reference to thefollowing Examples, but it should be understood that the presentinvention is not deemed to be limited thereto.

EXAMPLES Example 1 Materials and Methods Reagents and Proteins

The following reagents were obtained from the NIH AIDS Reference andReagent Program, Division of AIDS, NIAID: HIV-1_(BaL) gp120,HIV-1_(SF162) gp120, HIV-1_(CM235) gp120 from Protein Sciences,Corporation, HIV-1 gp120 Monoclonal Antibody (2G12) from Dr. HermannKatinger; HIV-1 gp120 Monoclonal Antibody (F105) from Dr. MarshallPosner and Dr. Lisa Cavacini. HIV-193_(MW959) gp120, HIV-1_(92UG21-9)gp120, HIV-1_(92US715) gp120 and SIV_(PBj2-8) HIV-1 gp120 and AN1 wereprovided by colleagues. Monoclonal Antibody 17b was obtained fromStrategic Biosolutions. CVN plasmid and anti-CVN polyclonal antibodieswere a gift from Biosyn Inc.

Example 2 Expression and Purification of CVN and CVN-12p1

BL21 Codon Plus (DE3) RIL competent cells (Stratagene) containing eitherCVN or the chimera were grown in Superbroth supplemented with 1 mMMgSO₄, 0.5% glucose and 25 μg/ml kanamycin in a 37° C. shaking incubatoruntil the absorbance at 600 nm read 1.2. Protein expression was inducedwith 1 mM IPTG for two hours at 37° C. The cells were then centrifugedat 3,000 rpm for 15 minutes and the resulting pellet was resuspended in100 mM Tris, 1 mM EDTA and 20% sucrose. This was mixed for one hour at4° C. followed by resuspension in cold water which was stirred overnightat 4° C. The lysates were centrifuged at 10,000 rpm for thirty minutesand the supernatant sterile filtered and dialyzed into 50 mM NaH₂PO₄,300 mM NaCl and 10 mM imidazole pH 8.0. The extracts were then adsorbedonto NiNTA agarose beads (Qiagen) and washed with three column volumesof 50 mM NaH₂PO₄, 300 mM NaCl and 20 mM imidazole pH 8.0. The proteinwas eluted with 50 mM NaH₂PO₄, 300 mM NaCl and 250 mM imidazole pH 8.0.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a4-20% polyacrylamide gel under reducing conditions (1%β-mercaptoethanol). Proteins were detected with Simply Blue CoomassieStain (Invitrogen). For immunoblot assays, proteins were transferred tonitrocellulose. The proteins were detected with a 1:10,000 dilution ofpolyclonal rabbit anti-CVN followed by anti-rabbit HRP. Images weredetected with chemilluminescent substrate (Amersham) and exposed tofilm. Proteins were quantified based on their absorbance at 280 nm.

Example 3 YU2 gp120 and sCD4 Production

Recombinant YU2-gp120 from HIV-1 was produced in Schneider 2 (S2)Drosophila cells under the control of the metallothionein promoter aspreviously described⁴¹. In brief, the cells were grown in Insect-Xpressmedium (Cambrex) supplemented with 10 mM L-glutamine and 1%antibiotic/antimycotic in shaker flasks at 28° C. Protein expression wasinduced with 750 μM copper sulfate until the viability as determined bytrypan blue was 70%. The supernatant was fractionated on an F105 mAbaffinity column, washed extensively and eluted with 100 mM glycine-HCl,pH 2.1. The samples were neutralized with 1M Tris, pH 8.0 bufferimmediately after elution. Fractions containing gp120 were pooled anddialyzed overnight at 4° C. against Dulbecco's phosphate bufferedsaline.

CHO-ST4.2 cells, which secrete the full extracellular domain of CD4,were obtained from Dr. Dan Littman through the AIDS Research andReference program Division of AIDS, NIAID. They were grown in a hollowfiber bioreactor (FiberCell Systems, Inc.) in HiQ CDM4CHO media(Hyclone) supplemented with 400 mM L-glutamine, 300 nM methotrexate and1% antibiotic/antimycotic. Supernatant from the CHO cells was diluted10-fold in cold 50 mM MES/50 mM NaCl pH 6.0 and passed through a 0.2 μMsterile filter. They were equilibrated with 50 mM MES/50 mM NaCl pH 6.0at 4° C., and fractions collected from a 50 mM MES/500 mM NaCl pH 6.0gradient. Fractions were dialyzed into 50 mM bis-tris propane pH 6.0,adjusted to pH 9.0 and loaded onto a Q-column equilibrated with bis-trispropane pH 9.0. The column flow through containing purified sCD4 waspooled and dialyzed into DPBS overnight at 4° C. All proteins wereanalyzed by SDS-PAGE/Coomassie stain to be of greater than 95% purity.

Example 4 Biacore Assays

All surface plasmon resonance studies were performed on a Biacore 3000(Biacore Inc., Uppsala, Sweden). CM5 dextran chip was derivatized byamine coupling with 250 RUs of YU2 gp120 or 2B6R (anti-IL5 receptorβchain) IgG as a control surface. Either CVN alone or the chimera waspassed over the surface at a flow rate of 30 μL/min, for two minutesfollowed by a four minute dissociation period. Surfaces were regeneratedwith multiple short pulses of 10 mM glycine HCl, pH 1.7.

Example 5 ELISA Assays

Ninety-six well plates (Corning) were coated with either 100 ng of theindicated gp120 or BSA overnight at 4° C. The wells were washed threetimes with 1×PBS-T (PBS, 0.1% Tween) and non-specific binding blockedwith 1% (wt/vol) BSA in PBS. This was followed by three washes with PBSTand incubation with the indicated doses of either CVN or the chimera forone hour at room temperature. The wells were washed and incubated with a1/1000 dilution of rabbit anti-CVN polyclonal antibody. After a one hourincubation, the wells were washed and incubated with a 1/2500 dilutionof anti-rabbit HRP (Amersham) for one hour at room temperature. Thewells were washed three times and the extent of binding was thendetermined by OPD (o-phenylenediamine, Sigma) and absorbance measured at450 nm.

Experiments were in triplicate and corrected for non-specific binding toa BSA surface.

Example 6

Antibody inhibition assays were performed as described above but witheither 1:20,000 dilution of F105, 50 μg/ml dilution of 2G12 or 2 μg/ml17b added to the CVN or chimera dilutions. The extent of CVN binding wasdetected with rabbit anti-CVN primary antibody. The data was plotted aspercent of binding in the absence of either CVN or the chimera.

For CD4 competition assays, 2 ng/μl YU2-gp120 or BSA was adsorbed onto a96 well plate. Serial dilutions of either CVN or the chimera were addedfor thirty minutes followed by the addition of 0.5 μg/ml sCD4 for thirtyminutes. The wells were washed and then incubated with anti-CD4(ARP356-NIBSC UK) for one hour at room temperature. This was followed bywashing and addition of anti-mouse HRP conjugated secondary antibody.The extent of binding was then determined by o-phenylenediamine (OPD)(Sigma) as described above.

Example 7 Viral Synergy Studies

Doubling dilutions of each drug (12p1 or CVN), alone or in fixedcombinations, were incubated with HIV-1 BaL (R5) for 1 h in 100 μlvolume prior to the addition of PM-1 cells (100 μl at 0.50×10⁶cells/ml). Dilutions and cells were in RPMI supplemented with 10% fetalcalf serum, pen, strep and glutamine. Cells were incubated for 7 daysand viral replication determined by gp120 ELISA. Each condition wasperformed in sextuplet and the mean used to calculate IC₅₀ values.Analysis of combined effects was also carried out using the medianeffect principle developed by Chou and Talalay^(39,40), and using thecomputer program CalcuSyn (BioSoft).

While the invention has been described in detail and with reference tospecific examples thereof, it will be apparent to one skilled in the artthat various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

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1. A chimeric protein comprising a first sequence coding for cyanovirin,a second sequence coding for 12p1 and a linker covalently connecting thefirst sequence coding for cyanovirin with the second sequence coding for12p1, wherein the first sequence coding for cyanovirin is selected fromthe group consisting of (a) at least nine contiguous amino acids of SEQID NO: 2, (b) nucleic acid sequence of SEQ ID NO: 1, (c) nucleic acidsequence of SEQ ID NO: 3, (d) amino acid sequence of SEQ ID NO: 4, and(e) nucleic acid sequence of SEQ ID NO: 5 and the second sequence codingfor 12p1 is selected from the group consisting of nucleic acid sequenceof SEQ ID NO: 6 and nucleic acid sequence of SEQ ID NO:
 7. 2. Thechimeric protein of claim 1, wherein the linker comprises nucleic acidsequence of SEQ ID NO:
 8. 3. The chimeric protein of claim 2, whereinthe linker has at least three repeats of SEQ ID NO:
 8. 4. The chimericprotein of claim 2, wherein the linker has at least five repeats of aSEQ ID NO. 8 or corresponds to SEQ ID NO:
 9. 5. The chimeric protein ofclaim 1 having a nucleotide sequence of SEQ ID NO:
 10. 6. The chimericprotein of claim 1 having a nucleotide sequence of SEQ ID NO:
 11. 7. Thechimeric protein of claim 3, wherein the linker has a nucleotidesequence of SEQ ID NO:
 7. 8. An HIV inhibitor comprising the chimericprotein of claim
 1. 9. A pharmaceutical composition comprising the HIVinhibitor of claim 1 and a pharmaceutically acceptable carrier.
 10. Apharmaceutical composition comprising: (i) an isolated and purifiedfirst nucleic acid molecule protein encoding a first sequence coding forcyanovirin, wherein the first sequence coding for cyanovirin is selectedfrom the group consisting of (a) at least nine contiguous amino acids ofSEQ ID NO: 2, (b) nucleic acid sequence of SEQ ID NO: 1, (c) nucleicacid sequence of SEQ ID NO: 3, (d) amino acid sequence of SEQ ID NO: 4,and (e) nucleic acid sequence of SEQ ID NO: 5; and (ii) an isolated andpurified second nucleic acid molecule protein encoding a second sequencecoding for 12p1, wherein the second sequence coding for 12p1 is selectedfrom the group consisting of nucleic acid sequence of SEQ ID NO: 6 andnucleic acid sequence of SEQ ID NO: 7, wherein said pharmaceuticalcomposition is effective to prevent, treat or alleviate an HIV infectionin a mammal.
 11. A method of preventing or alleviating an HIV infection,the method comprising providing the pharmaceutical composition of claim10 in an amount effective to prevent, treat or alleviate an HIVinfection in a mammal.
 12. A method of preventing or alleviating an HIVinfection, the method comprising providing the pharmaceuticalcomposition of claim 11 in an amount effective to prevent, treat oralleviate an HIV infection in a mammal.
 13. A vector comprising thechimeric protein of claim
 1. 14. A host cell containing the vector ofclaim
 13. 15. A method of producing a protein, which method comprisesexpressing a protein in a host cell of claim
 14. 16. The host cell ofclaim 14, wherein said host cell is an autologous or a homologousmammalian cell.
 17. The host cell of claim 14, wherein said host cell isa nonpathogenic bacterium or a nonpathogenic yeast.
 18. The host cell ofclaim 14, wherein said host cell is a lactobacillus.
 19. An antibody tothe chimeric protein of claim 1.